molecules
Article
The Use of Mass Spectrometric Techniques to
Differentiate Isobaric and Isomeric Flavonoid
Conjugates from Axyris amaranthoides
Łukasz Marczak 1, *, Paulina Znajdek-Awiżeń 2 and Wiesława Bylka 2
1
2
*
European Centre for Bioinformatics and Genomics, Institute of Bioorganic Chemistry,
Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
Department of Pharmacognosy, Poznan University of Medical Sciences, Świecickiego 4,
60-781 Poznan, Poland; [email protected] (P.Z.-A.); [email protected] (W.B.)
Correspondence: [email protected]; Tel.: +48-061-852-8503; Fax: +48-061-852-0532
Academic Editors: Arturo San Feliciano and Celestino Santos-Buelga
Received: 27 July 2016; Accepted: 8 September 2016; Published: 19 September 2016
Abstract: Flavonoids are a group of compounds that are commonly found in various plants, where
they play important roles in many processes, including free radical scavenging and UV protection.
These compounds can also act as chemical messengers, physiological regulators or protectants
against pathogens in the defense reactions of plants. Flavonoid activity is regulated by the addition
of various substituents, usually mono- or oligosaccharides of common sugars, such as glucose,
rhamnose or galactose. In some plants, glucuronic acid is attached, and this sugar is often acylated by
phenylpropanoic acids. Identification of these compounds and their derivatives is of great importance
to understanding their role in plant metabolism and defense mechanisms; this research is important
because flavonoids are frequently a significant constituent of the human diet. In this study, we identify
the flavonoid conjugates present in Axyris amaranthoides L. extracts and demonstrate the usefulness of
high-resolution mass spectrometry (HRMS) analyzers for the differentiation of isobaric compounds
and the utility of fragmentation spectra for the differentiation of isomeric structures. According to
our knowledge, some of the structures, especially dehydrodiferulated conjugates of tricin, whose
structures are proposed here have been found for the first time in plant material.
Keywords: collision-induced dissociation; electrospray; flavonoid conjugates; Axyris amaranthoides;
tandem mass spectrometry; fragmentation spectra; isomeric compounds; isobaric compounds
1. Introduction
Flavonoids are present in plants mainly as glycosides; glucuronides, in which uronic acids occur
as sugar units, are less frequent [1]. The role of glucuronides in plants has not been yet clarified.
It is speculated that they can provide protection against herbivores [2]. Flavonoid glucuronidation can
also be catalyzed by enzymatic reactions in human and animal metabolic pathways [3].
The main flavonoid glucuronides identified thus far in plants include glucuronoflavones,
especially apigenin and luteolin, as well as tricin, chrysoeriol, scutellarein and the glucuronoflavonols
quercetin, kaempferol, isorhamnetin and myricetin [4]. These constituents have been reported
in Asteraceae (Cirsium rivulare, Erigeron multiradiatus, Tanacetum parthenium) [5–7], Polygonaceae
(Polygonum amphibium, P. aviculare) [1,8], Lamiaceae (Scutellaria indica, S. baicalensis, Salvia officinalis,
S. palestina [9–12], Scrophulariaceae (Picria fel-terrae) [13], Pedaliaceae (Uncarina sp.) [14] and
Chenopodiaceae (Spinacia oleracea) [15]. Diglucuronoflavones identified in a few species from
Fabaceae (Medicago truncatula, M. radiata, M. sativa) [16–19], Lamiaceae (Perilla ocimoides) [20],
Poaceae (Secale cereale) [21] and Hydrocharitaceae (Elodea canadensis) [22] are particularly rare.
Molecules 2016, 21, 1229; doi:10.3390/molecules21091229
www.mdpi.com/journal/molecules
Molecules 2016, 21, 1229
2 of 15
In Medicago sp., isolated diglucuronoflavones were additionally acylated with the hydroxycinnamic
acids caffeic, ferulic, sinapic and p-coumaric acids [17–19].
Some glucoronidic derivatives of flavonoids have been shown to exert antioxidant and
anti-inflammatory activities [8,23]. In in vitro studies, quercetin 3-O-glucuronide prevented vascular
smooth muscle cell hypertrophy by angiotensin II. Moreover, flavonol glucuronides from
Polygonum amphibium L. induced apoptosis in HL-60 and Jurkat cells [1]. A study conducted in
rabbits demonstrated that a flavonoid glucuronide fraction isolated from Tulipa gesneriana exerted
microvascular protective effects against skin vascular permeability induced by chloroform or
histamine [24].
Axyris amaranthoides L. (Chenopodiaceae) is a herbaceous annual plant (20–80 cm in height),
commonly known as Russian Pigweed. This plant originated in Asia but is naturally widespread
throughout the USA. A. amaranthoides herb is used in Chinese folk medicine as a gastrointestinal
spasmolytic and to regenerate the liver, as well as alleviating rheumatic pain and swelling [25,26].
Utilization of liquid chromatography and mass spectrometry in analysis of phenolic compounds,
especially flavonoids, is extensively studied in the literature and the use of different mass spectrometric
analyzers and ionization techniques have grown to be the most common method in this type
of studies [27,28]. High resolution tandem mass spectrometry (MS/MS) systems are capable to
deliver precise information on molecular weight of compounds, and collision induced dissociation
(CID) fragmentation used together with soft ionization techniques provides additional structural
information [29]. Moreover, analysis of samples in both positive and negative ionization modes
may support the study due to different fragmentation patterns of protonated and deprotonated
molecules [30]. Nevertheless, all this information is not sufficient for complete structural elucidation
of studied compounds due to almost identical fragmentation pathways of positional isomers.
This situation is in accordance with flavonoid derivative fragmentation, where sugar moieties can be
substituted at different positions of aglycones; moreover, these sugar moieties are often also acylated
with organic acids at different hydroxyl groups [27]. There are some proposals in literature trying to
find a method to distinguish differentially substituted isomeric flavonoid molecules. One of them
is measuring relative abundances of specific fragmentation ions [31], and the other is electrospray
ionization (ESI) analysis of metal complexes [32]. Such approaches are interesting but do not allow
unambiguous identification and depend strongly on analysis parameters and interpretation skills.
The only confident way for complex structural analysis is still the use of different complementary
spectroscopic methods for elucidation of molecule structure. In most cases, information derived
from NMR and MS analysis leads to unambiguous structure elucidation [17,18]. Such approaches
are possible only when compound of interest is present in relatively high concentrations and can be
purified from the complex mixtures. Another way to confirm proper compound identification is the
use of purified or commercially available standards. This way, it is possible to compare retention times
of standards with potentially identified compound as well as comparison of their MS and multiple
MS/MS (MSn) spectra. This approach is also commonly used in combination with ultraviolet diode
array detectors (UV-DAD) [33]. High resolution MS was used to identify saponins and flavonoids in
Camellia oleifera [34], using a ultra high performance liquid chromatography coupled with electrospray
ionization hybrid linear trap quadrupole orbitrap mass spectrometry (UHPLC-LTQ-orbitrap-MSn)
system, it was possible to tentatively characterize seven flavonol glycosides, and MS3 spectra were used
for unambiguous aglycone characterization. Another interesting work showing usefulness of the high
performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MSn) approach
for comprehensive plant metabolites characterization was published by Wang and co-workers [35],
where authors propose general strategies for analysis of complex samples. Application of such a
strategy allowed temporary identification of 48 flavonoid derivatives among 144 compounds in total,
while NMR analysis was further used for structures confirmation of some novel compounds.
Based on a literature search, A. amaranthoides has been the subject of just several phytochemical
studies. Extracts of A. amaranthoides roots and seeds were found to contain large amounts of
Molecules 2016, 21, 1229
3 of 15
phytoecdysteroids, and 1α,20R-dihydroxyecdysone, 20E-hydroxyecdysone (polypodine A) and
5,20E-dihydroxyecdysone (polypodine B) have been isolated [36,37]. There is no further information
in the available literature on flavonoid content of A. amaranthoides that we could confront with our
results. This facts are making analysis on MS systems more difficult due to lack of information on
generalMolecules
glycosylation
pattern of aglycones provided by enzymatic systems responsible for
2016, 21, 1229
3 of acylation
15
and glucosidation of flavonoids specifically in A. amaranthoides. Therefore, all proposed structures in
5,20E-dihydroxyecdysone (polypodine B) have been isolated [36,37]. There is no further information
this paper should be treated as tentative and are based on the most common substitution patterns
in the available literature on flavonoid content of A. amaranthoides that we could confront with our
found inresults.
available
literature.
This facts
are making analysis on MS systems more difficult due to lack of information on
In this
study,
we demonstrate
A. amaranthoides
herb is a rich
source
of flavonoid
compounds,
general
glycosylation
pattern ofthat
aglycones
provided by enzymatic
systems
responsible
for acylation
glucosidation
of flavonoids specifically
in A.quite
amaranthoides.
allthe
proposed
structures
in
mainly and
tricin
acylated glucuronides,
which are
rare in Therefore,
plants. To
best of
our knowledge,
this
paper
should
be
treated
as
tentative
and
are
based
on
the
most
common
substitution
patterns
some of the structures proposed here have been shown for the first time in plants.
found in available literature.
In this
study, we demonstrate that A. amaranthoides herb is a rich source of flavonoid compounds,
2. Results and
Discussion
mainly tricin acylated glucuronides, which are quite rare in plants. To the best of our knowledge,
some ofGlycosides
the structures
proposed
here have been shown for the first time in plants.
2.1. Flavonoid
of Axyris
amaranthoides
2. Results
and Discussion
Due
to the presence
in examined fractions of a large number of flavonoids of similar structures
and polarities, the chemical constituents in the mixture were identified using HPLC combined with
2.1. Flavonoid Glycosides of Axyris amaranthoides
high-resolution tandem quadrupole-time of flight (QToF) mass spectrometry. A detailed analysis of
the presence
in the
examined
fractions
of a large numberofof35
flavonoids
of similar
structures
the MS andDue
MS2tospectra
led to
successful
characterization
flavonoid
conjugates,
including
and polarities, the chemical constituents in the mixture were identified using HPLC combined with
previously identified compounds [19] and previously unknown flavonoid derivatives.
high-resolution tandem quadrupole-time of flight (QToF) mass spectrometry. A detailed analysis of
Allthe
ofMS
theand
tentatively
identified compounds are presented in Table 1. Most of them were
MS2 spectra led to the successful characterization of 35 flavonoid conjugates, including
tricin derivatives
(18
compounds).
Others
compounds
included
derivatives
of chrysoeriol (seven
previously identified compounds [19]
and previously
unknown
flavonoid
derivatives.
compounds),
(five
compounds),
apigenin
(two compounds),
(one tricin
compound)
Allisorhamnetin
of the tentatively
identified
compounds
are presented
in Table 1. Mostluteolin
of them were
derivatives
(18compound).
compounds).Assigning
Others compounds
included
derivatives
of chrysoeriol
(seven due to
and syringetin
(one
the correct
structures
for aglycones
was possible
isorhamnetin
(five compounds),
apigenin
(two compounds),
luteolin
compound)
pseudo compounds),
MS3 analysis
and comparison
of obtained
fragmentation
spectra
with(one
spectra
of standards
and syringetin (one compound). Assigning the correct structures for aglycones was possible due to
and spectra found
in
literature
or
deposited
in
common
spectral
libraries.
This
work
was done as
pseudo MS3 analysis and comparison of obtained fragmentation spectra with spectra of standards
described
in
our
earlier
papers
[19,38].
Structures
of
identified
aglycones
are
presented
inas
Figure 1,
and spectra found in literature or deposited in common spectral libraries. This work was done
and fragmentation
spectra
proposed
structures
of the conjugates
arepresented
shown in
described in our
earlierand
papers
[19,38]. Structures
of identified
aglycones are
in supplementary
Figure 1,
and fragmentation
spectra
and proposed
structures
the conjugates
are shown in flavonoid
supplementary
data (Figures
S1–S35). The
conjugates
included
both of
acylated
and non-acylated
glycosides
data (Figures
S1–S35).
The and/or
conjugates
included both
acylated
and non-acylated
glycosides
with attached
glucose,
xylose
glucuronic
acid
moieties.
Most of theflavonoid
recognized
metabolites
with attached glucose, xylose and/or glucuronic acid moieties. Most of the recognized metabolites
were acylated
with various phenolic acids, 13 metabolites were feruloylated, nine metabolites were
were acylated with various phenolic acids, 13 metabolites were feruloylated, nine metabolites were
acylatedacylated
with p-coumaric
acid, and one metabolite was acylated with sinapic acid. Additionally,
with p-coumaric acid, and one metabolite was acylated with sinapic acid. Additionally, we
we propose
thatthat
twotwo
of of
thethemetabolites
acylatedwith
with
dehydrodiferulic
and
one
was acylated
propose
metabolites were
were acylated
dehydrodiferulic
acid, acid,
and one
was
acylated
with benzoic
acid. acid.
with benzoic
Figure 1. Structures of flavonoid aglycones identified in Axyris amaranthoides.
Figure 1. Structures of flavonoid aglycones identified in Axyris amaranthoides.
Molecules 2016, 21, 1229
4 of 15
Table 1. Flavonoid glycosides tentatively identified in Axyris amaranthoides.
#
Name
Elemental
Composition
Exact Monoisotopic
Mass (Da)
Fragment Ions in
Positive Ion Mode (m/z)
Fragment Ions in
Negative Ion Mode (m/z)
1
Isorhamnetin 3(7)-O-glucuronopyranosyl-(1-2)-O-glucuronopyranoside
C28 H28 O19
668.1224
669, 493, 317
667, 351, 315, 193, 175
2
Chrysoeriol 7-O-glucuronopyranosyl-(1-2)-O-glucuronopyranoside
C28 H28 O18
652.1275
653, 477, 301
651, 351, 299, 175
3
Chrysoeriol 7-O-[2-O-feruloyl-glucuronopyranosyl-(1-3)-O-glucopyranoside]
C38 H38 O20
814.1956
815, 653, 477
813, 513
4
Luteolin 7-O-[20 -O-feruloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C37 H34 O21
814.1592
815, 463,2 87
814, 527, 285, 193
5
Tricin 7-O-glucuronopyranosyl-(1-2)-O-glucuronopyranoside
C29 H30 O19
682.1381
683, 507, 331
681, 351, 193, 175
6
Tricin 7-O-[2-O-feruloyl-glucopyranosyl-(1-3)-O-glucuronopyranoside]
C39 H40 O21
844.2062
845, 683, 507
843, 513
7
Tricin 7-O-{20 -O-feruloyl-[glucopyranosyl-(1-30 )-O-glucopyranosyl]-(1-2)-Oglucuronopyranoside}
C45 H50 O26
1006.259
1007, 507
1005, 675, 513
8
Isorhamnetin 3(7)-O-glucuronopyranoside
C22 H20 O13
492.0904
493, 317
491, 315
3(7)-O-{30 -O-coumaroyl-[glucuronopyranosyl-(1-20 )-O-glucopyranosyl]-
9
Isorhamnetin
(1-2)-O-glucuronopyranoside}
C43 H44 O26
976.212
977, 815, 493, 317
975, 811, 659, 513, 495, 337,
315
10
Syringetin 3-O-[20 -O-feuroyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C39 H38 O23
874.1804
875, 523, 347
873, 527, 333, 193
7-O-{20 -O-feruloyl-[glucopyranosyl-(1-30 )]-O-glucuronopyranosyl-(1-2)-O-
11
Apigenin
glucuronopyranoside}
C43 H44 O25
960.2171
961, 799, 447, 271
959, 689, 527, 193
12
Isorhamnetin 3(7)-O-[20 -O-feruloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C38 H36 O22
844.1698
845, 493, 317, 177
843, 649, 527, 315, 193
C44 H46 O26
990.2277
991, 829, 477, 301
989, 689, 495
13
7-O-{30 -O-feruloyl-[glucuronopyranosyl-(1-20 )]-O-glucopyranosyl-(1-2)-O-
Chrysoeriol
glucuronopyranoside}
7-O-{30 -O-feruloyl-[glucuronopyranosyl-(1-2)]-O-glucopyranosyl-(1-2)-O-
14
Tricin
glucuronopyranoside}
C45 H48 O27
1020.238
1021, 859, 507, 331
1019, 689, 495
15
Tricin 7-O-{30 -O-coumaroyl-[glucuronopyranosyl-(1-20 )]-O-glucopyranosyl-(1-2)-Oglucuronopyranoside}
C44 H46 O26
990.2277
991, 829, 507, 331
989, 659, 495
16
Tricin 7-O-glucuronopyranoside
C23 H22 O13
506.105
507, 331
505, 490, 329, 314
C40 H40 O23
888.196
889, 507, 383, 331, 207
887, 557, 399, 223
827, 527, 333, 193
7-O-[20 -O-sinapoyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
17
Tricin
18
Chrysoeriol 7-O-[20 -O-feruloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C38 H36 O21
828.1749
829, 477, 353, 301, 177
19
Tricin 7-O-[20 -O-feruloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C39 H38 O22
858.1854
859, 507, 331
857, 527, 193
20
Tricin 7-O-[20 -O-coumaroyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C38 H36 O21
828.1749
829, 507, 331, 147
827, 497, 333, 163
21
Tricin 7-O-{3-O-feruloyl-[glucopyranosyl-(1-20 )-O-glucuronopyranosyl]-(1-2)-Oglucuronopyranoside}
C45 H48 O27
1020.238
1021, 683, 507, 339, 177
1019, 689, 495
22
Tricin 5-O-glucuronopyranosyl-7-O-glucopyranoside
C29 H32 O18
668.1588
669, 507, 331
667, 491, 329
23
Tricin 7-O-[20 -O-5-hydroxyferuloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C39 H38 O23
874.1803
875, 507, 369, 331
873, 543, 351, 209
24
Chrysoeriol 40 -O-xylopyranosyl-7-O-[20 -O-coumaroyl-glucuronopyranosyl-(1-2)-Oglucuronopyranoside]
C42 H42 O24
930.2066
931, 799, 609, 433, 301
929, 797, 497, 163
Molecules 2016, 21, 1229
5 of 15
Table 1. Cont.
Elemental
Composition
Exact Monoisotopic
Mass (Da)
Fragment Ions in
Positive Ion Mode (m/z)
Fragment Ions in
Negative Ion Mode (m/z)
Tricin 40 -O-xylopyranosyl-7-O-[20 -O-coumaroyl-glucuronopyranosyl-(1-2)-Oglucuronopyranoside]
C43 H44 O25
960.2171
961, 829, 507, 463, 331
959, 827, 497
26
Tricin 7-O-[glucuronopyranosyl-(1-2)-O-methyloglucuronopyranoside]
C30 H32 O19
696.1537
697, 521, 331
695, 533, 329
27
Isorhamnetin 3(7)-O-[20 -O-coumaroyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C37 H34 O21
814.1592
815, 493, 317
813, 649, 497, 315, 163
28
Chrysoeriol 7-O-glucuronopyranoside
C22 H20 O12
476.0954
477, 301
-
29
Apigenin 7-O-[20 -O-feruloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C37 H34 O20
798.1643
799, 447, 331, 271
797, 603, 527, 333, 193
30
Chrysoeriol 7-O-[20 -O-coumaroyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C37 H34 O20
798.1643
799, 477, 301
797, 497
31
Tricin
7-O-[20 -O-feruloyl-glucuronopyranosyl-(1-2)-O-methyloglucuronopyranoside]
C40 H40 O22
872.2011
873, 521, 331, 177
871, 677, 519, 329
32
Tricin 7-O-[20 -O-benzoyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C36 H34 O20
786.1643
787, 507, 331
785, 455, 333, 121
33
Tricin 7-O-[20 -O-coumaroyl-methyloglucuronopyranosyl-(1-2)-O-glucuronopyranoside]
C39 H38 O21
842.1905
843, 507, 331
841, 695, 633, 511, 329, 163
34
Tricin 7-O-[20 -O-dehydrodiferuloyl-glucopyranosyl-(1-2)-O-glucuronopyranoside]
C49 H48 O25
1036.2479
1037, 531, 331
1035, 705, 547, 371
C49 H48 O26
1052.2428
1053, 859, 507, 331
1051, 721, 563, 387
#
25
35
Name
Tricin
7-O-[20 -O-dehydrodiferuloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
Molecules 2016, 21, 1229
6 of 15
Because we exclusively used MS techniques to identify the mixture components, we were
unable to assign the positions of particular moieties in aglycone molecules, and we were also
unable to distinguish the exact sugar substituents. Based only on our experience and the available
literature,
we2016,
propose
Molecules
21, 1229 tentative structures of identified compounds. We are aware that
6 of analysis
15
of standards should be done to perform correct confirmation of our statements; nevertheless,
Because
we exclusively
MS techniques
to identify
mixture
components,
we were
unable
we dealt with
complex
mixturesused
of compounds
often
found the
in very
low
concentrations,
and
additional
to assign the positions of particular moieties in aglycone molecules, and we were also unable to
separation of specific compounds or even chemical synthesis would be impossible or very complicated
distinguish the exact sugar substituents. Based only on our experience and the available literature,
and time-consuming.
Many
studies
described
the typical
patterns
of flavonoids.
we propose tentative
structures
of have
identified
compounds.
We are substitution
aware that analysis
of standards
NMR studies
have
identified
the
phenylpropenoic
acidic
moieties
(p-coumaric,
ferulic
should be done to perform correct confirmation of our statements; nevertheless, we dealtand
withsinapic
acids) of
these mixtures
conjugates,
and the positions
of in
thevery
ester
bonds
and the glycosidic
bonds
between the
complex
of compounds
often found
low
concentrations,
and additional
separation
of specific
or even chemical
synthesis
would be
impossible
or veryreports
complicated
and timeaglycone
and thecompounds
sugar moiety(ies)
have been
established
[16].
In addition,
of similar
structures
consuming.
Many
studies
have
described
the
typical
substitution
patterns
of
flavonoids.
have been confirmed in various species [17,18]. It should be noted here that, thanks to useNMR
of the high
studies have identified the phenylpropenoic acidic moieties (p-coumaric, ferulic and sinapic acids) of
resolution
mass spectrometry (HRMS) system, we were able to distinguish isobaric fragment losses
these conjugates, and the positions of the ester bonds and the glycosidic bonds between the aglycone
of ferulate
moiety from glucuronate moiety (loss of 176.046 Da to 176.031 Da), and coumarate from
and the sugar moiety(ies) have been established [16]. In addition, reports of similar structures have
rhamnose
moiety
(loss
146.036
Da to
146.057
Da). Additionally,
thesethanks
isobaric
substituents
been confirmed
in of
various
species
[17,18].
It should
be noted here that,
to use
of the highcan be
differentiated
based
comparison
of positive
negative
iondistinguish
fragmentation
since
ferulate
resolution
masson
spectrometry
(HRMS)
system,and
we were
able to
isobaricspectra,
fragment
losses
ferulate moietyneutral
from glucuronate
moiety
(loss coumarate
of 176.046 Dagives
to 176.031
Da),
from
gives aofcharacteristic
loss of 194
Da and
a loss
of and
164 coumarate
Da. Based
on these
rhamnose
moietycertain
(loss of structures
146.036 Da to
Da). Additionally,
isobaric
substituents
can be with
findings,
we propose
for146.057
the flavone
derivativesthese
found
in Axyris
amaranthoides
differentiated
based
on
comparison
of
positive
and
negative
ion
fragmentation
spectra,
since
ferulate
the reservation that these structures were not determined directly using analytical methods that permit
gives a characteristic neutral loss of 194 Da and coumarate gives a loss of 164 Da. Based on these
unambiguous determination.
findings, we propose certain structures for the flavone derivatives found in Axyris amaranthoides with
Inthe
thisreservation
study, allthat
samples
analyzed
in both
positive
and negative
ion modes,
spectra
these were
structures
were not
determined
directly
using analytical
methods
that were
acquired
using
a
high-resolution
q-ToF
mass
spectrometer
that
enabled
precise
measurements
of
permit unambiguous determination.
+ and [M − H]− ions in both MS and MS2 modes. Because we have
m/z valuesInfor
[M all
+ H]
thisall
study,
samples
were analyzed in both positive and negative ion modes, spectra were
acquired
using a high-resolution
q-ToF
mass spectrometer
enabled
precise
m/z
previously
discussed
the necessity for
high-resolution
MSthat
systems
[19],
heremeasurements
we will onlyofemphasize
+ and [M − H]− ions in both MS and MS2 modes. Because we have previously
values
for
all
[M
+
H]
the inability of low-resolution MS analyzers such as quadrupole or ion trap instruments to determine
the necessity for high-resolution MS systems [19], here we will only emphasize the inability
isobaricdiscussed
compounds
or isobaric fragment ions. Furthermore, knowing the exact mass of protonated or
of low-resolution MS analyzers such as quadrupole or ion trap instruments to determine isobaric
deprotonated ions and having precisely calibrated the system prior to analysis enhances the ability of
compounds or isobaric fragment ions. Furthermore, knowing the exact mass of protonated or
these methods
to correctly
determine
structures.
Finally,
the prior
use of
UPLC enhances
system greatly
affected
deprotonated
ions and having
precisely
calibrated
the system
to aanalysis
the ability
the acquisition
of the data;
chromatographic
resolution
wasthe
anuse
important
the analysis
of these methods
to correctly
determine structures.
Finally,
of a UPLCfactor
systemingreatly
affected due to
the close
of some of theresolution
flavone glucuronides.
It factor
is notable
the due
resolving
the structural
acquisition similarities
of the data; chromatographic
was an important
in the that
analysis
closesystems
structural
similarities
some ofcompounds
the flavone glucuronides.
It is within
notable 8that
theduring
resolving
power to
of the
UPLC
enabled
the of
studied
to be separated
min
a 12-min
power
of
UPLC
systems
enabled
the
studied
compounds
to
be
separated
within
8
min
during
a
12chromatographic run. Hyphenation of this system with a fast high-resolution mass spectrometer
min chromatographic run. Hyphenation of this system with a fast high-resolution mass spectrometer
allowed the acquisition of MS2 spectra
for consecutive compounds. Figure 2 shows the extracted ion
allowed the acquisition of MS2 spectra for consecutive compounds. Figure 2 shows the extracted ion
chromatogram
(EIC)
for
the
product
ion
at m/z 815; seven peaks were observed, three of which were
chromatogram (EIC) for the product ion at m/z 815; seven peaks were observed, three of which were
successfully
characterized
based
onon
their
andfragmentation
fragmentation
spectra
(Figure
successfully
characterized
based
theirexact
exactmass
mass and
spectra
(Figure
3). 3).
Figure 2. Extracted ion chromatogram at m/z 815 showing all isomeric and isobaric ions. Asterisks
Figure 2. Extracted ion chromatogram at m/z 815 showing all isomeric and isobaric ions. Asterisks
indicate isomeric compounds not possible to distinguish due to very similar spectra to compounds 3,
indicate isomeric compounds not possible to distinguish due to very similar spectra to compounds 3,
4 or 27.
4 or 27.
Molecules 2016, 21, 1229
Molecules 2016, 21, 1229
7 of 15
7 of 15
Figure 3. Mass spectra registered in positive and negative ion mode for isomeric and isobaric
Figure 3. compounds:
Mass spectra
registered in positive and negative ion mode for isomeric and
chrysoeriol 7-O-[2-O-feruloyl-glucuronopyranosyl-(1-3)-O-glucopyranoside] (3),
isobaric compounds:
chrysoeriol
7-O-[2-O-feruloyl-glucuronopyranosyl-(1-3)-O-glucopyranoside]
(3),
luteolin 7-O-[2′-O-feruloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
(4) and isorhamnetin
0
3-O-[2′-O-coumaroyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside]
(27).
luteolin 7-O-[2 -O-feruloyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside] (4) and isorhamnetin
3-O-[20 -O-coumaroyl-glucuronopyranosyl-(1-2)-O-glucuronopyranoside] (27).
Each of the compounds has a different aglycone, i.e., chrysoeriol (3), luteolin (4) and
isorhamnetin (27). Furthermore, compounds 4 and 27 are isomers. In addition to containing different
these two conjugates
differently.
Compound
is conjugated with
acid, (4) and
Each aglycones,
of the compounds
has are
a acylated
different
aglycone,
i.e., 4chrysoeriol
(3),ferulic
luteolin
and (27).
compound
27 is conjugated
with coumaric
thus,
these two
have
the same different
isorhamnetin
Furthermore,
compounds
4 andacid;
27 are
isomers.
Incompounds
addition to
containing
molecular formula. These acylating moieties were differentiated using positive and negative ion
aglycones,mode
these
two conjugates are acylated differently. Compound 4 is conjugated with ferulic
fragmentation spectra. The mass of compound 3 differs from those of compounds 4 and 27 by
acid, and compound
2740ismDa,
conjugated
with
acid;
thus,
thesetotwo
compounds
have
approximately
which is too
largecoumaric
of a difference
to be
attributed
improper
calibration;
we the same
it hasThese
a different
molecularmoieties
formula sowere
that these
results suggestusing
that compound
isobaric
molecular propose
formula.
acylating
differentiated
positive3 isand
negative ion
with compounds
4 and 27
(seemass
Table of
1). compound
Again, analysis
positive
and those
negativeofspectra
leads to 4 and 27
mode fragmentation
spectra.
The
3 of
differs
from
compounds
unambiguous identification of the substituents in compound 3. Other isomeric compounds were
by approximately
40 mDa, which is too large of a difference to be attributed to improper calibration;
differentiated in our study by analyzing fragmentation spectra, including the following compound
we propose
it has
a different
molecular
that15,these
results
that
compound 3 is
pairs:
29 and
30, 18 and 20,
10 and 23,formula
11 and 25, so
13 and
and 14
and 21.suggest
Other pairs
of isobaric
compounds
that were
effectively
extractsanalysis
of Axyris of
amaranthoides
are 1negative
and 22, and
6
isobaric with
compounds
4 and
27 (seerecognized
Table 1).inAgain,
positive and
spectra
leads
and 12. identification of the substituents in compound 3. Other isomeric compounds were
to unambiguous
differentiated in our study by analyzing fragmentation spectra, including the following compound
pairs: 29 and 30, 18 and 20, 10 and 23, 11 and 25, 13 and 15, and 14 and 21. Other pairs of isobaric
compounds that were effectively recognized in extracts of Axyris amaranthoides are 1 and 22, and 6
and 12.
2.2. Fragmentation Pathways of Flavone Glycoconjugates
Only eight out of the 35 compounds detected in Axyris amaranthoides were non-acylated flavone
mono- and diglycosides. These compounds contained linear saccharides, which were attached most
Molecules 2016, 21, 1229
8 of 15
probably at position 7 of chrysoeriol and tricin. Based on previously reported work [39,40], the most
probable site of glycosylation is position 3 of isorhamnetin, but based only on MS/MS spectra, we can
not exclude the possibility that the saccharides are attached at position 7 as well. Compounds 8, 16 and
28 were mono-glucuronates of isorhamnetin, tricin and chrysoeriol, respectively. The glycosidic moiety
of compounds 1, 2 and 5 comprised two combined glucuronic acid molecules, and compound 22 was
glucuronosylo-glucoside but with two sugar moieties substituted at different positions. Compound 26
contained one methyl ester of glucuronic acid.
CID fragmentation spectra of these compounds, which were acquired in positive ion mode,
included only product ions that were created after successive cleavages of glycosidic bonds between
sugars and between the sugar moiety and the aglycone (see Supplementary Materials). A positive
charge was retained on the aglycone, and an entire series of Yn + product ions were observed [41,42].
This fragmentation pattern is equivalent to that observed earlier by us [19] and to patterns observed
by others for all flavonoid glycosides [42].
The fragmentation pathway observed in the negative ion mode (deprotonation) was different
from the fragmentation pathway observed in the positive ion mode. As in our previous work [19,27],
we observed a predominant loss of aglycones in the case of diglycosides, and the charge was retained
on the sugar moiety, representing the most abundant peak in the spectrum. For diglucuronides,
we observed an ion at m/z 351, which is typical of these compounds, as described in our previous
study [19]. Interestingly, for monoglucosides, the fragmentation pathway observed in the negative ion
mode is similar to that observed with positive spectra; namely, we observed a loss of sugar and charge
retention on the aglycone, which suggests that only di- and triglucuronate moieties can efficiently
stabilize the negative charge. This finding is consistent with other reports [43]. In the positive ion
mode, a loss of hexose was observed from compound 22, followed by the loss of glucuronic acid;
however, in the negative ion mode, the opposite was true, i.e., glucuronic acid was lost before hexose.
This clearly indicates that the two substituents are directly attached to the aglycone in different
positions; therefore, it is not possible that these two sugar moieties are linked to each other. In this
case, analysis of mass spectra does not give the information on exact position of glucuronic acid with
respect to glucose moiety.
The glycosylated flavone tricin 7-O-[glucuronopyranosyl-(1-2)-O-methylglucuronopyranoside]
(compound 26) was also recognized. When compared to compound 5, which is a non-methylated
tricin diglucuronide, we observed a completely different fragmentation scheme. In the methylated
diglucuronide, charge is retained on an aglycone fragment in the negative mode; therefore,
MS/MS spectra recorded for 26 using the positive and negative ion modes appear very similar.
This demonstrates that although two carboxyl groups in the diglucuronate moiety strongly stabilize the
product ion [19], blocking one of these carboxyl groups dramatically alters the stability of this negative
ion, resulting in charge retention on the aglycone. Few studies have analyzed flavone diglucuronides
using negative-ion MS/MS, although many quercetin and methylquercetin diglucuronides have been
isolated from animal tissues or body fluids [44,45]. In these reports, no product ion at m/z 351 was
reported, suggesting either that quercetin glucuronides act differently or that both sugars were attached
to different hydroxyl groups on the aglycone. However, the cited authors did not address this issue.
2.3. Fragmentation Pathways of Acylated Flavone Glycoconjugates
Among the compounds found in this work, 27 were acylated with various phenolic acids, mainly
ferulic acid (16 compounds) and p-coumaric acid (nine compounds), and with sinapic, benzoic
and hydroxyferulic acids (one compound each). In addition, we found two tricin conjugates,
which we propose are acylated with dehydrodiferulic acids. All of the characterized flavonoid
conjugates contained at least two sugar moieties, and some contained three. We detected no acylated
monoglycosylated flavones, consistent with our previous report [19] and those of others [16–18].
Analysis of the fragmentation pathways of acylated flavones showed that they behave similarly
to non-acylated compounds, especially in the positive ion mode, for which we observed a sequential
Molecules 2016, 21, 1229
Molecules 2016, 21, 1229
9 of 15
9 of 15
of the
fragmentation
of acylated
flavones
showed
that they
behave similarly
loss of Analysis
consecutive
substituents
thatpathways
left the aglycone
ion
as a final
fragment.
Apparently,
terminal
to
non-acylated
compounds,
especially
in
the
positive
ion
mode,
for
which
we
observed
sequential
phenolic acid groups always leave the pseudomolecular ion together with the sugar unitsato
which they
loss of consecutive substituents that left the aglycone ion as a final fragment. Apparently, terminal
are connected; consequently, we observed characteristic losses of 352 Da for ferulate and 322 Da for
phenolic acid groups always leave the pseudomolecular ion together with the sugar units to which
coumarate. The same effect was observed with hydroxyferulate (368 Da) and benzoate (280 Da).
they are connected; consequently, we observed characteristic losses of 352 Da for ferulate and 322 Da
However, because only one of each type of these compounds (23 and 32) was observed in our
for coumarate. The same effect was observed with hydroxyferulate (368 Da) and benzoate (280 Da).
experiment, we cannot be certain that this result is common in all such cases.
However, because only one of each type of these compounds (23 and 32) was observed in our
When phenylpropenoic acid units were attached to non-terminal sugar moieties, we observed
experiment, we cannot be certain that this result is common in all such cases.
different
fragmentation
behavior:
for compounds
11, 13, 14 and
in which
middle
When
phenylpropenoic
acid units
were attached7,to9,non-terminal
sugar15,
moieties,
we the
observed
glucuronates
were
acylated,
we
observed
the
loss
of
a
single
sugar
unit
(in
all
cases,
this
sugar
different fragmentation behavior: for compounds 7, 9, 11, 13, 14 and 15, in which the middle
was
a
hexose,
presumably
glucose
(162
Da)).
Subsequently,
an
acylated
glucuronide
fragment
glucuronates were acylated, we observed the loss of a single sugar unit (in all cases, this sugar was was
a
removed
the resulting
residue, and the
final fragmentation
step was was
cleavage
of the
hexose,from
presumably
glucoseprotonated
(162 Da)). Subsequently,
an acylated
glucuronide fragment
removed
O-glycosidic
bond between
glucuronic
acid
and
aglycone.
For compound
we assigned
from the resulting
protonated
residue,
and
thethe
final
fragmentation
step was21,cleavage
of thethe
acylation
position
to
the
glucuronate
unit
that
was
directly
attached
to
tricin.
The
initial
fragmentation
O-glycosidic bond between glucuronic acid and the aglycone. For compound 21, we assigned the
step
was the position
elimination
moiety
Da); this
step was
followed
the loss
acylation
to of
thea glucuronate-glucose
glucuronate unit that
was (352
directly
attached
to tricin.
Thebyinitial
was the elimination
of a glucuronate-glucose
moiety
(352 Da);onthis
was
of fragmentation
a ferulate unit. step
Surprisingly,
in the final fragmentation
step, a charge
was retained
thestep
glucuronic
followed
by
the
loss
of
a
ferulate
unit.
Surprisingly,
in
the
final
fragmentation
step,
a
charge
was
acid residue, which is not typical of positive ion mode fragmentation.
retained
on
the
glucuronic
acid
residue,
which
is
not
typical
of
positive
ion
mode
fragmentation.
The fragmentation spectra of acylated glycoconjugates acquired in the negative ion mode were
The fragmentation
spectra offragment
acylated ions
glycoconjugates
acquired in
the negative
were
interesting;
we observed common
having characteristic
masses
of m/z ion
497,mode
m/z 527
and
interesting;
we
observed
common
fragment
ions
having
characteristic
masses
of
m/z
497,
m/z
527
and
m/z 513. These masses correspond to acylated sugar moieties comprising two molecules of glucuronic
m/zor513.
These masses
sugar
moieties
comprising
two molecules
glucuronic
acid
glucuronic
acid correspond
conjugatedtotoacylated
a glucose
unit.
We have
previously
describedof
fragmentation
acid or glucuronic acid conjugated to a glucose unit. We have previously described fragmentation
pathways leading to these fragments [19]. Here, we report a further characteristic ion at m/z 495,
pathways leading to these fragments [19]. Here, we report a further characteristic ion at m/z 495,
which corresponds to a linear fragment comprising two units of glucuronic acid and one unit of
which corresponds to a linear fragment comprising two units of glucuronic acid and one unit of
glucose (Figure 4a). This fragment was detected in the spectra obtained for compounds 9, 13, 14, 15
glucose (Figure 4a). This fragment was detected in the spectra obtained for compounds 9, 13, 14, 15
and 21 and appears to be present only when sugar rings are connected by glycosidic bonds in positions
and 21 and appears to be present only when sugar rings are connected by glycosidic bonds in
1 → 2. We did not observe this ion in compounds 7 and 11, which also contain linear triglucosides, but
positions 1 → 2. We did not observe this ion in compounds 7 and 11, which also contain linear
instead
observed
a glycosidic
bond ina positions
→ 3 in
between
the1two
(seethe
thetwo
Supplementary
triglucosides,
but
instead observed
glycosidic1bond
positions
→ 3units
between
units (see
Materials).
Another Materials).
observed ion
that was
not previously
is a fragment
511 that
the Supplementary
Another
observed
ion that wasreported
not previously
reportedof
is m/z
a fragment
was
observed
in
the
negative
ion
spectrum
of
compound
33;
we
assigned
this
result
to
a
fragment
of m/z 511 that was observed in the negative ion spectrum of compound 33; we assigned this result
that
fromoriginated
p-coumaric
acid
and twoacid
units
of two
glucuronic
acid, in which
of these
to originated
a fragment that
from
p-coumaric
and
units of glucuronic
acid,one
in which
oneunits
of
contained
a methylated
group
(Figure
4b).(Figure 4b).
these units
contained acarboxyl
methylated
carboxyl
group
Figure
Structuresof
ofthe
the characteristic
characteristic product
flavonoid
glucuronates,
(a) m/z
in
Figure
4. 4.Structures
productions
ionsofof
flavonoid
glucuronates,
(a) 495
m/z found
495 found
acylated
at
middle
sugar
moiety
compounds,
with
linear
fragment
comprising
two
units
of
glucuronic
in acylated at middle sugar moiety compounds, with linear fragment comprising two units of
acid and one unit of glucose; and (b) m/z 511 corresponding to methylated diglucuronate acylated
glucuronic
acid and one unit of glucose; and (b) m/z 511 corresponding to methylated diglucuronate
with p-coumaric acid. Ions were found in the collision induced dissociation fragmentation (CID
acylated with p-coumaric acid. Ions were found in the collision induced dissociation fragmentation
MS/MS) spectra, registered in negative mode, referred to the compounds from Table 1 and spectra in
(CID MS/MS) spectra, registered in negative mode, referred to the compounds from Table 1 and spectra
supplementary data.
in supplementary data.
Molecules 2016, 21, 1229
10 of 15
As was demonstrated above by the results obtained for compound 22, the analysis of spectra
that were acquired in both positive and negative ion modes resulted in the tentative identification of
structures with sugar moieties substituted at two positions. This was also observed with the acylated
compounds 24 and 25. In the positive ion spectrum, we observed two possible losses occurring
from the pseudomolecular ion: the loss of a fragment (132 Da) corresponding to a pentose molecule
(presumably xylose) and a second possible cleavage that can be attributed to the loss of coumaroyl
glucuronide (322 Da). Further fragmentation steps form a deprotonated chrysoeriol molecule.
In addition, in the negative ion spectrum, we observed the loss of a neutral pentose fragment, followed
by cleavage of an aglycone, to a characteristic ion at m/z 497, representing a deprotonated coumaroyl
diglucuronide [19]. The same fragmentation pathways were observed with compound 25 except that a
different aglycone was present—in this case, tricin. It should be noted here that it was not possible to
define the position of xylose substituent based only on the mass spectra, but we can presume that, due
to steric hindrance, the most possible site for xylose group in compounds 24 and 25 is hydroxyl group
at 40 position.
In this work, we detected fine fragmentation spectra of precursors with relatively high molecular
masses exceeding 1000 Da; i.e., compounds 34 and 35, with masses of 1036 Da and 1052 Da, respectively.
Analysis of the fragmentation pathways led to the conclusion that these compounds were tricin
glycoconjugates that were acylated with dehydrodiferulic acids; to the best of our knowledge,
these flavone derivatives are reported for the first time. We observed that the acylating group
in compound 35 is cyclobutane diferulic acid [46,47]. This ferulic acid dimer is formed by
cyclodimerization in plant exposed to UV irradiation [48]. Therefore, we cannot be certain if this
compound is native to Axyris amaranthoides or if it was produced during sample preparation when
the samples were subjected to UV rays from daylight irradiation. This compound has a characteristic
fragmentation pattern in the positive ion mode. In the initial fragmentation step, one molecule of
ferulic acid is detached from its dimer; consequently, an ion at m/z 859 is created that corresponds
to a molecule containing ferulated diglucuronide (compound 19). Further fragmentation steps are
identical to those observed with compound 19. In the negative ion mode, we observed a classic loss of
aglycone, followed by the consecutive loss of two glucuronide moieties, resulting in the formation
of an ion (m/z 371) that corresponds to a deprotonated cyclobutane dimer of ferulic acid. Finally,
a fragment at m/z 193 is present corresponding to single, deprotonated molecule of ferulic acid.
Compound 34 contains non-cyclic dehydrodiferulic acid, which is frequently found in plant
cell walls, especially in grasses, where it serves as a chemical link between chains of arabinoxylans,
among others [47,49]. Such dimers are produced by enzymatic reactions involving oxidative enzymes
such as peroxidase; thus, it is likely that compound 34 is a native constituent of these plants.
Dehydrodiferulates occur naturally in many isomeric forms [50]; therefore, we cannot conclude which
isoform is present in our finding. The fragmentation behavior of these components is not typical and is
different from that observed for compound 35. Here, in positive mode, we detected the loss of a 506 Da
fragment corresponding to aglycone glucuronide (330 Da for tricin and 176 Da for glucuronic acid).
Charge is retained on dehydrodiferuloyl glucoside (m/z 531), although the aglycone ion itself remains
present, and this ion is the second most abundant ion in the spectrum. The fragmentation pathway
obtained by analyzing the negative ion mode spectrum provides supplementary information regarding
the identified compound. In the initial fragmentation step, we observed a typical loss of aglycone,
followed by the loss of glucuronic acid; then, dehydrodiferulate is cleaved, followed by the loss of
ferulic acid (176 Da). Consequently, a [M − H]− ion with m/z 371 is observed, which corresponds to a
glucose α-hydroxyferulate fragment. The proposed fragmentation scheme is presented in Figure 5.
Molecules 2016, 21, 1229
Molecules 2016, 21, 1229
11 of 15
11 of 15
Figure
5. Proposed
fragmentation
pathwayfor
forcompound
compound 34
spectrum
registered
Figure
5. Proposed
fragmentation
pathway
34based
basedononMS/MS
MS/MS
spectrum
registered
in negative ion mode.
in negative ion mode.
3. Experimental
3. Experimental Section
3.1. General
3.1. General
Thin-layer chromatography (TLC) was performed on cellulose plates (Merck, Darmstadt,
Thin-layer
chromatography
(TLC)
was performed
cellulose
(Merck,
Darmstadt,
Germany) using
1% KOH in MeOH
for detection.
The plateson
were
irradiatedplates
with UV
light at 366
nm
Germany)
1%spraying.
KOH in MeOH for detection. The plates were irradiated with UV light at
before using
and after
Column
366 nm before
andchromatography
after spraying. was performed using Sephadex LH-20 (Pharmacia Biotech AB,
Uppsala,
Sweden)
and cellulose
powder Whatman
CF 11 (Whatman
Ltd., Maidstone,
Column chromatography
was performed
using Sephadex
LH-20 International
(Pharmacia Biotech
AB, Uppsala,
Kent,
UK).
The
solvent
systems
used
were
as
follows:
S1:
(EtOAc–MeOH–H
2O 100:6:6); S2: (EtOAc–
Sweden) and cellulose powder Whatman CF 11 (Whatman International Ltd., Maidstone, Kent, UK).
MeOH–H2O 100:10:9.5); S3: (EtOAc–MeOH–H2O: 100:16:13) and S3: (EtOAc–MeOH–H2O: 100:18:15).
The solvent
systems used were as follows: S1: (EtOAc–MeOH–H2 O 100:6:6); S2: (EtOAc–MeOH–H2 O
Solid-phase extraction (SPE) was performed using a System BAKER SPE 12-G) equipped with a
100:10:9.5); S3: (EtOAc–MeOH–H2 O: 100:16:13) and S3: (EtOAc–MeOH–H2 O: 100:18:15).
Bond Elut C18 cartridge (Agilent Technologies, Palo Alto, CA, USA). Methanol, ethyl acetate and
Solid-phase
extraction (SPE) was performed using a System BAKER SPE 12-G) equipped with
KOH were obtained from POCh, Gliwice, Poland. All reagents and solvents used for chromatography
a Bond
Elut
C18 cartridge
(Agilent Technologies,
Palo Alto, CA, USA). Methanol, ethyl acetate and
were
of analytical
or spectrometric
grade.
KOH were obtained from POCh, Gliwice, Poland. All reagents and solvents used for chromatography
Plant Materials
were 3.2.
of analytical
or spectrometric grade.
Axyris amaranthoides herb was cultivated in the garden of the Department of Medical Plants at
Poznan University of Medical Sciences. The seeds were obtained from Warsaw University Botanical
Garden.amaranthoides
The plant washerb
collected
August 2012
during
the flowering
period and of
authenticated
by at
Axyris
wasin
cultivated
in the
garden
of the Department
Medical Plants
MScUniversity
Katarzyna Guranowska
from Warsaw
VoucherUniversity
specimens were
Poznan
of Medical Sciences.
The University,
seeds wereWarszawa,
obtained Poland.
from Warsaw
Botanical
deposited
in thewas
Department
Poznanthe
University
of Medical
Sciences,
Poznan, by
Garden.
The plant
collectedofinPharmacognosy,
August 2012 during
flowering
period and
authenticated
Poland (AX 253).
3.2. Plant Materials
MSc Katarzyna Guranowska from Warsaw University, Warszawa, Poland. Voucher specimens were
deposited in the Department of Pharmacognosy, Poznan University of Medical Sciences, Poznan,
Poland (AX 253).
3.3. Extraction of Phenolic Compounds
Air-dried A. amaranthoides herb (300 g) was successively extracted with MeOH and 50% MeOH.
The combined extracts were evaporated to dryness, and the dry extract was treated with hot H2 O,
Molecules 2016, 21, 1229
12 of 15
filtered and then partitioned between CHCl3 and EtOAc. The concentrated chloroform extract was
loaded onto a Sephadex LH-20 column (Pharmacia Biotech AB, Uppsala, Sweden) and washed with
50% MeOH to yield a crude powder (10 mg). The ethyl acetate fraction was chromatographed on
cellulose powder with S1 as the eluent to yield a crude product (12 mg), which was then purified by
Sephadex LH-20 column chromatography and eluted with 50% MeOH. The aqueous residue after
extraction with CHCl3 and EtOAc was separated on a cellulose column; fractions were eluted first with
S2, then with S3 and finally, with S4, yielding 128 fractions. After TLC, control fractions 73–90 were
combined and rechromatographed on Sephadex LH-20 using first H2 O and then 50% MeOH to yield
another product (30 mg). Final purification of the obtained products was achieved using solid-phase
extraction (SPE) according to the Svedström method with minor modifications [51]. The cartridge was
first conditioned with 10 mL of methanol and 5 mL of water; then, 5 mL of water was added to the dry
compounds, and 1 mL of this solution was loaded onto the cartridge. Fractions were then eluted with
water followed by mixtures of H2 O:MeOH of increasing concentration from 5% MeOH to 40% MeOH.
3.4. Liquid Chromatography with Mass Spectrometric Detection
All fractions (see Section 3.3) were analyzed using an HPLC/MS system comprising a Waters
Acquity UPLC system (Waters, Milford, MA, USA) combined with a Q-ToF mass spectrometer,
model micrOTOF-q (Bruker Daltonics, Bremen, Germany). Analyses were carried out using Zorbax
Eclipse XDB-C18 columns (2.1 mm × 150 mm, 3.5 µm and 2.1 mm × 100 mm, 1.8 µm; Agilent).
Chromatographic separation using the Waters Acquity UPLC system was performed at a flow rate
of 0.6 mL/min using mixtures of the following two solvents: A (99.5% H2 O, 0.5% formic acid (v/v));
B (99.5% acetonitrile, 0.5% formic acid (v/v)). The column effluent was split 3:2 to allow 0.2 mL/min
to be delivered to an ESI ion source. The elution steps were as follows: 0–8 min linear gradient from
5% to 30% B, 8–10 min linear gradient to 95% B, 10–12 min of isocratic flow at 95% B and a return to
the initial condition after 3 min.
The micrOTOF-Q spectrometer consisted of ESI operating at ±4.5 kV, nebulization with nitrogen
at 1.6 bar and a dry gas flow of 8.0 L/min at a temperature of 220 ◦ C. For the pseudo MS3 analyses,
additionally, in-source induced dissociation (ISCID) was set up at 80 eV to cleave O-glycosidic
bonds in flavonoid conjugates enabling aglycone fragmentation spectra acquisition. The system
was calibrated externally using a calibration mixture containing sodium formate clusters. Additional
internal calibrations were performed for every run by injecting the calibration mixture using a divert
valve during LC separation. All calculations were performed using the high precision calibration
(HPC) quadratic algorithm. These calibrations yielded an accuracy of less than 10 ppm. MS/MS
spectra were acquired at a frequency of one scan per second for ions that were chosen on the basis
of preliminary MS experiments. The collision energy was dependent on the molecular masses of
the compounds and was set between 10 and 25 eV. The instrument operated at a resolution higher
than 10,000 (FWHM, full width at half maximum) using the program micrOTOF control version 2.3,
and data were analyzed using the Data Analysis version 4 package, which was supplied by Bruker.
Metabolite profiles were registered in positive and negative ion modes.
4. Conclusions
The use of an LC-MS/MS system comprising a rapid HPLC and a tandem MS/MS spectrometer
equipped with a high-resolution time-of-flight analyzer permits the structural characterization of
flavonoid glycoconjugates containing glucuronic acid and phenolic acyl substituents. However, due to
the availability of complementary information resulting from differences in the fragmentation patterns
for the positive and negative ions of these compounds, it is essential that analyses are performed using
both modes of ionization. It is of particular importance that mechanisms observed for glycosides
containing more than one molecule of glucuronic acid are dramatically different from those of flavonoid
glycoconjugates containing sugars without carboxyl groups, especially when using the negative ion
mode. Ions presented in Figure 4 may serve as diagnostic ones for correct recognition of presented
Molecules 2016, 21, 1229
13 of 15
here specific groups of flavonoid diglucuronides. HRMS analyses performed in both positive and
negative ion modes might permit the structural characterization of glucuronyloglucosides and di- or
triglucuronides. Such a system allows also for unambiguous differentiation between isomeric and
isobaric compounds in opposite to low resolution MS. Additionally, we showed that, in MS/MS mode,
differentiation of isobaric fragments (ferulate and glucuronate, coumarate and rhamnose, etc.) is also
of great importance and is possible only on high-resolution MS systems. The use of modern analytical
equipment might enable the characterization of potential new molecules as presented in this study,
in which we demonstrate the acylation of flavone glucuronides with dehydrodiferulic acids. However,
one should keep in mind that mass spectrometry itself does not allow for unambiguous identification
of compounds and proposed structures should always be additionally confirmed using other methods,
such as NMR spectroscopy. In our case, unfortunately, it was not possible due to the fact that these
compounds were present in samples in very low amounts.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
9/1229/s1.
Acknowledgments: The authors wish to thank Maciej Stobiecki for critical reading of the manuscript and valuable
comments on the discussion of results.
Author Contributions: Ł.M., P.Z.-A. and W.B. conceived and designed the experiments; P.Z.-A. and W.B. prepared
plant material and performed extractions; Ł.M. performed LC/MS analyses, Ł.M., P.Z.-A. and W.B. analyzed the
data; and Ł.M., P.Z.-A. and W.B wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Smolarz, H.D.; Budzianowski, J.; Bogucka-Kocka, A.; Kocki, J.; Mendyk, E. Flavonoid glucuronides with
anti-leukaemic activity from Polygonum amphibium L. Phytochem. Anal. 2008, 19, 506–513. [CrossRef] [PubMed]
Poe, M.L.; Bates, A.; Onyilagha, J. Distribution of Leaf Flavonoid Aglycones and Glucuronides in the Genus
Phaseolus and Related Genera. Int. J. Biol. 2013, 5, 36–43. [CrossRef]
Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C.; Jiménez, L. Polyphenols: Food sources and bioavailability.
Am. J. Clin. Nutr. 2004, 79, 727–747. [PubMed]
Andersen, O.M.; Markham, K.R. Flavonoids. Chemistry, Biochemistry and Applications; CRC, Taylor & Francis:
Boca Raton, FL, USA, 2006.
Nazaruk, J.; Gudej, J. Flavonoid compounds from the flowers of Cirsium rivulare (Jacq.) All. Acta Pol. Pharm.
Drug Res. 2003, 1, 87–89.
Zhang, Z.; Liu, Y.; Luo, P.; Zhang, H. Separation and Purification of Two Flavone Glucuronides from Erigeron
multiradiatus (Lindl.) Benth with Macroporous Resins. J. Biomed. Biotechnol. 2009, 2009. [CrossRef] [PubMed]
Williams, C.A.; Harborne, J.B.; Geiger, H.; Robin, J.; Hoult, S. The flavonoids of Tanacetum parthenium and
T-vulgare and their anti-inflammatory properties. Phytochemistry 1999, 51, 417–423. [CrossRef]
Granica, S.; Czerwińska, M.E.; Zyzyńska-Granica, B.; Kiss, A.K. Antioxidant and anti-inflammatory flavonol
glucuronides from Polygonum aviculare L. Fitoterapia 2013, 91, 180–188. [CrossRef] [PubMed]
Tomimori, T.; Miyaichi, Y.; Imoto, Y.; Kizu, H.; Suzuki, C. Studies on the constituents of Scutellaria species.
IV. On the flavonoid constituents of the root of Scutellaria baicalensis Georgi (4). Yakugaku Zasshi 1984, 104,
529–534. [PubMed]
Liu, G.; Rajesh, N.; Wang, X.; Zhang, M.; Wu, Q.; Li, S.; Chen, B.; Yao, S. Identification of flavonoids in the
stems and leaves of Scutellaria baicalensis Georgi. J. Chromatogr. B 2011, 879, 13–14. [CrossRef] [PubMed]
Lu, Y.; Yeap Foo, L. Flavonoid and phenolic glycosides from Salvia officinalis. Phytochemistry 2000, 55, 263–267.
[CrossRef]
Al-Qudah, M.A.; Al-Jaber, H.I.; Abu Zarga, M.H.; Abu Orabi, S.T. Flavonoid and phenolic compounds from
Salvia palaestina L. growing wild in Jordan and their antioxidant activities. Phytochemistry 2014, 99, 115–120.
[CrossRef] [PubMed]
Huang, Y.; De Bruyne, T.; Apers, S.; Ma, Y.; Claeys, M.; Pieters, L.; Vlietinck, A. Flavonoid glucuronides from
Picria fel-terrae. Phytochemistry 1999, 52, 1701–1703. [CrossRef]
Molecules 2016, 21, 1229
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
14 of 15
Yamazaki, K.; Iwashina, T.; Kitajima, J.; Gamou, Y.; Yoshida, A.; Tannowa, T. External and internal flavonoids
from Madagascarian Uncarina species (Pedaliaceae). Biochem. Syst. Ecol. 2007, 35, 743–749. [CrossRef]
Aritomi, M.; Kawasaki, T. Three highly oxygenated flavone glucuronides in leaves of Spinacia oleracea.
Phytochemistry 1984, 23, 2043–2047. [CrossRef]
Kowalska, I.; Stochmal, A.; Kapusta, I.; Janda, B.; Pizza, C.; Piacente, S.; Oleszek, W. Flavonoids from barrel
medic (Medicago truncatula) aerial parts. J. Agric. Food Chem. 2007, 55, 2645–2652. [CrossRef] [PubMed]
Stochmal, A.; Piacente, S.; Pizza, C.; De Riccardis, F.; Leitz, R.; Oleszek, W. Alfalfa (Medicago sativa L.)
Flavonoids. 1. Apigenin and luteolin glycosides from aerial parts. J. Agric. Food Chem. 2001, 49, 753–758.
[CrossRef] [PubMed]
Stochmal, A.; Simonet, A.M.; Macias, F.A.; Oleszek, W. Alfalfa (Medicago sativa L.) flavonoids. 2. Tricin and
chrysoeriol glycosides from aerial parts. J. Agric. Food Chem. 2001, 49, 5310–5314. [CrossRef] [PubMed]
Marczak, L.; Stobiecki, M.; Jasinski, M.; Oleszek, W.; Kachlicki, P. Fragmentation pathways of acylated
flavonoid diglucuronides from leaves of Medicago truncatula. Phytochem. Anal. 2010, 21, 224–233. [CrossRef]
[PubMed]
Yoshida, K.; Kameda, K.; Kondot, T. Diglucuronoflavones from purple leaves of Perilla ocimoides.
Phytochemistry 1993, 33, 917–919. [CrossRef]
Schulz, M.; Strack, D.; Weissenböck, G.; Markham, K.R.; Dellamonica, G.; Chopin, J. Two luteolin
O-glucuronides from primary leaves of Secale cereale. Phytochemistry 1985, 24, 343–345. [CrossRef]
Mues, R. Species Specific Flavone Glucuronides in Elodea Species. Biochem. Syst. Ecol. 1983, 11, 261–265.
[CrossRef]
Pawlak, K.; Bylka, W.; Jazurek, B.; Matlawska, I.; Sikorska, M.; Manikowski, H.; Bialek-Bylka, G.
Antioxidant activity of flavonoids of different polarity, assayed by modified ABTS cation radical
decolorization and EPR technique. Acta Biol. Crac. Ser. Bot. 2010, 52, 97–104. [CrossRef]
Budzianowski, J.; Korzeniowska, K.; Chmara, E.; Mrozikiewicz, A. Microvascular protective activity of
flavonoid glucuronides fraction from Tulipa gesneriana. Phytother. Res. 1999, 13, 166–168. [CrossRef]
Wu, Z.Y.; Raven, P.H.; Hong, D.Y. Flora of China. Vol. 5 (Ulmaceae through Basellaceae); Science Press: Beijing,
China, 2003; Missouri Botanical Garden Press: St. Louis, MO, USA, 2003.
Aswal, B.S.; Bhakuni, D.S.; Goel, A.K.; Kar, K.; Mehrotra, B.N. Screening of Indian plants for biological
activity—Part XI. Indian J. Exp. Biol. 1984, 22, 487–504. [PubMed]
Stobiecki, M. Applications of separation techniques hyphenated to mass spectrometer for metabolic profiling.
Curr. Org. Chem. 2001, 5, 335–349. [CrossRef]
March, R.E.; Miao, X.S.; Metcalfe, C.D.; Stobiecki, M.; Marczak, L. A fragmentation study of an isoflavone
glycoside, genistein-7-O-glucoside, using electrospray quadrupole time-of-flight mass spectrometry at high
mass resolution. Int. J. Mass Spectrom. 2004, 232, 171–183. [CrossRef]
Shao, Y.; Jiang, J.; Ran, L.; Lu, C.; Wei, C.; Wang, Y. Analysis of Flavonoids and Hydroxycinnamic Acid
Derivatives in Rapeseeds (Brassica napus L. var. napus) by HPLC-PDA-ESI(−)-MSn/HRMS. J. Agric.
Food Chem. 2014, 62, 2935–2945. [CrossRef] [PubMed]
Wojakowska, A.; Perkowski, J.; Góral, T.; Stobiecki, M. Structural characterization of flavonoid glycosides
from leaves of wheat (Triticum aestivum L.) using LC/MS/MS profiling of the target compounds.
J. Mass Spectrom. 2013, 48, 329–339. [CrossRef] [PubMed]
Abad-García, B.; Garmón-Lobato, S.; Berrueta, L.A.; Gallo, B.; Vicente, F. Practical guidelines for
characterization of O-diglycosyl flavonoid isomers by triple quadrupole MS and their applications for
identification of some fruit juices flavonoids. J. Mass Spectrom. 2009, 44, 1017–1025. [CrossRef] [PubMed]
Pikulski, M.; Brodbelt, J.S. Differentiation of flavonoid glycoside isomers by using metal complexation and
electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 2003, 14, 1437–1453. [CrossRef]
[PubMed]
Lv, Y.-W.; Hu, W.; Wang, Y.-L.; Huang, L.-F.; He, Y.-B.; Xie, X.-Z. Identification and Determination of
Flavonoids in Astragali Radix by High Performance Liquid Chromatography Coupled with DAD and
ESI-MS Detection. Molecules 2011, 16, 2293–2303. [CrossRef] [PubMed]
Feng, M.; Zhu, Z.; Zuo, L.; Chen, L.; Yuan, Q.; Shan, G.; Luo, S.-Z. A strategy for rapid
structural characterization of saponins and flavonoids from the testa of Camellia oleifera Abel seeds by
ultra-high-pressure liquid chromatography combined with electrospray ionization linear ion trap-orbitrap
mass spectrometry. Anal. Methods 2015, 7, 5942–5953. [CrossRef]
Molecules 2016, 21, 1229
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
15 of 15
Wang, S.; Chen, L.; Leng, J.; Chen, P.; Fan, X.; Cheng, Y. Fragment ion diagnostic strategies for the
comprehensive identification of chemical profile of Gui-Zhi-Tang by integrating high-resolution MS,
multiple-stage MS and UV information. J. Pharm. Biomed. Anal. 2014, 98, 22–35. [CrossRef] [PubMed]
Sarker, S.D.; Sik, V.; Rees, H.H.; Dinan, L. 1α,20R-dihydroxyecdysone from Axyris amaranthoides.
Phytochemistry 1998, 49, 2305–2310. [CrossRef]
Dinan, L. Distribution of phytoecdysteroids within Chenopodiaceae. Eur. J. Entomol. 1995, 92, 295–300.
Wojakowska, A.; Piasecka, A.; García-López, P.M.; Zamora-Natera, F.; Krajewski, P.; Marczak, Ł.; Kachlicki, P.;
Stobiecki, M. Structural analysis and profiling of phenolic secondary metabolites of Mexican lupine species
using LC-MS techniques. Phytochemistry 2013, 92, 71–86. [CrossRef] [PubMed]
Antunes-Ricardo, M.; Moreno-García, B.E.; Gutiérrez-Uribe, J.A.; Aráiz-Hernández, D.; Alvarez, M.M.;
Serna-Saldivar, S.O. Induction of Apoptosis in Colon Cancer Cells Treated with Isorhamnetin Glycosides
from Opuntia Ficus-indica Pads. Plant Foods Hum. Nutr. 2014, 69, 331–336. [CrossRef] [PubMed]
Sikorska, M.; Matlawska, I. Kaempferol, isorhamnetin and their glycosides in the flowers of Asclepias syriaca L.
Acta Pol. Pharm. Drug Res. 2001, 58, 269–272.
Domon, B.; Costello, C.E. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS
spectra of glycoconjugates. Glycoconj. J. 1988, 5, 397–409. [CrossRef]
Vukics, V.; Guttman, A. Structural characterization of flavonoid glycosides by multi-stage mass spectrometry.
Mass Spectrom. Rev. 2008, 29. [CrossRef] [PubMed]
Kachlicki, P.; Einhorn, J.; Muth, D.; Kerhoas, L.; Stobiecki, M. Evaluation of glycosylation and malonylation
patterns in flavonoid glycosides during LC/MS/MS metabolite profiling. J. Mass Spectrom. 2008, 43, 572–586.
[CrossRef] [PubMed]
Lee, W.J.; Choi, S.W. Quantitative changes of polyphenolic compounds in mulberry (Morus alba L.) leaves in
relation to varieties, harvest period, and heat processing. Prev. Nutr. Food Sci. 2012, 17, 280–285. [CrossRef]
[PubMed]
Mullen, W.; Hartley, R.C.; Crozier, A. Detection and identification of 14 C-labelled flavonol metabolites by
high-performance liquid chromatography-radiocounting and tandem mass spectrometry. J. Chromatogr. A
2003, 1007, 21–29. [CrossRef]
Dobberstein, D.; Bunzel, M. Identification of ferulate oligomers from corn stover. J. Sci. Food Agric. 2010, 90,
1802–1810. [CrossRef] [PubMed]
Renger, A.; Steinhart, H. Ferulic acid dehydrodimers as structural elements in cereal dietary fibre. Eur. Food
Res. Technol. 2000, 211, 422–428. [CrossRef]
Ford, W.C.; Hartley, D.R. GC/MS characterization of cyclodimers from p-coumaric and ferulic acids by
photodimerization—A possible factor influencing cell wall biodegradability. J. Sci. Food Agric. 1989, 46,
301–310. [CrossRef]
Micard, V.; Grabber, J.H.; Ralph, J.; Renard, C.M.G.C.; Thibault, J.F. Dehydrodiferulic acids from sugar-beet
pulp. Phytochemistry 1997, 44, 1365–1368. [CrossRef]
Stobiecki, M.; Busko, M.; Marczak, L.; Bednarek, P.; Pislewska, M.; Wojtaszek, P. The complexity of oxidative
cross-linking of phenylpropanoids: Evidence from an in vitro model system. Funct. Plant Biol. 2002, 29,
853–864. [CrossRef]
Svedström, U.; Vuorela, H.; Kostiainen, R.; Laakso, I.; Hiltunen, R. Fractionation of polyphenols in
hawthorn into polymeric procyanidins, phenolic acids and flavonoids prior to high-performance liquid
chromatographic analysis. J. Chromatogr. A 2006, 1112, 103–111. [CrossRef] [PubMed]
Sample Availability: Not availaible.
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